Angiogenesis is a regulated process involving the formation of new blood vessels. It plays an essential role in normal growth, embryonic development, wound healing, and other physiological processes (Yancopoulos et al. (2000) Nature 407(6801):242-8). Moreover, de novo angiogenesis is involved in several disease states including cancer, where the formation of new “embryonic-like” blood vessels (referred to as neovascularization herein) appear that differ from normal vasculature with regards to structure and function (Hanahan and Weinberg (2000) Cell 100(1):57-70). A number of in vivo and in vitro studies have demonstrated biological differences between normal and disease-associated vasculature as determined using various model systems of angiogenesis offering the ability to develop novel anti-angiogenic compounds that can selectively inhibit vessel formation of the embryonic-type, tumor-associated endothelial-type for therapy of neovascular disease. In light of these opportunities for therapy, an intense search for potential targets that can specifically inhibit tumor and other neovascular disease-associated endothelial cell growth and function is ongoing. In an attempt to identify such targets, strategies have been designed to identify cell surface antigens of tumor stroma as well as isolate specific proteins or RNA that are expressed in neovascular endothelial cells (Rettig et al. (1992) Proc Natl. Acad Sci USA 89(22):10832-6; St. Croix et al. (2000) Science 289: 1197-1202). These strategies have identified a cell surface protein that appears to be specifically expressed in neovascular endothelial cells called endosialin (or tumor endothelial marker 1, TEM1). The use of antibodies in immunohistochemistry studies of malignant tissues have found good expression of endosialin in a number of neovascular endothelial cells in malignant tissues, tumor stromal cells, and putative endothelial leukocytes [(Brady et al. (2004) Human endosialin (tumor endothelial marker 1) is abundantly expressed in highly malignant and invasive brain tumors. J. Neuropathol Exp Neurol. 63:1274-83; Rettig et al. (1992); Conejo-Garcia et al. (2005) Vascular leukocytes contribute to tumor vascularization. Blood 105:679-81)] while expression in cell lines derived from embryonic-like endothelial cultures such as but not limited to HUVEC (Human Umbilical Vein Endothelial Cells) or HMVEC-(Neonatal Dermal Microvascular Endothelial Cells) is limited (available online through Cambrex Corp.). Analysis of antibodies that can bind to endosialin have identified a set of cells that express this antigen in endothelial cell cultures as well as a subset of dendritic-like cells in normal tissue of patients. Recently it has been shown that leukocytes contain cells that can form endothelial cells in vessels of tumors, supporting the use of antibodies in targeting these endosialin-positive cell types for therapy (Peters et al. (2005) Contribution of bone marrow-derived endothelial cells to human tumor vasculature. Nat. Med. 11:261-262).
Neovascularization is associated with a number of disease states. In cancer it is believed that neovascularization is important to supply tumors with blood. In non-oncology diseases such as retinopathy and macular degeneration, uncontrolled neovascularization causes loss of sight (Wilkinson-Berka (2004) Curr Pharm Des. 10(27):3331-48; Das and McGuire (2003) Prog Retin Eye Res. 22(6):721-48). Moreover, several reports have identified a role of neovascularization in inflammatory disease (Paleolog and Miotla (1998) Angiogenesis 2(4):295-307). Methods to better define the embryonic-like endothelial and precursor cells associated with these disease states will lead to the development of novel drugs to treat these diseases. The development of antibodies that can specifically target endothelial cells associated with cancer or neovascular disease (age-related macular degeneration, retinopathy, inflammation, etc.) offers the ability to treat these diseases.
A difficult problem in effective antiangiogenic and proangiogenic therapy is the inability to specifically target and kill or suppress the action of these cells via pharmaceutical compounds. An approach to get better specificity to treat cancer or neovascular disease is the use of antibodies that can target specific antigens expressed in cancer or neo-endothelial cells or precursors that are not expressed or are expressed at a lower level on normal cells. These targets can be exploited using antibodies to kill antigen-bearing cells by inhibiting the biological activity of the antigen, eliciting an immune effector function by complement-dependent cytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity (ADCC); or by delivering immuno- or radio-conjugates that, when delivered to the antigen-bearing cells, specifically kill the target cell. Finding antibodies that can specifically bind to and effectively kill antigen-bearing cells involved in tumorigenesis and neovascular-associated disease, such as inflammation, age-related macular degeneration, and retinopathy, has proven difficult for many biological compounds (Dhanabal (2005) Anti-angiogenic therapy as a cancer treatment paradigm Curr Med Chem Anti-Canc Agents 5:115-30). This has been due in part to the inability to get robust killing due to lack of immune-effector activity or lack of efficient internalization of antibodies carrying immunotoxins.
In 1992, Rettig et al. described monoclonal antibodies that recognize antigens on vessels within various cancer types (Rettig et al. (1992) Proc Natl. Acad Sci USA 89(22):10832-6). One of these was designated FB5, which recognizes a ˜100 kDa protein on the surface of a neuroblastoma cell line, LA1-5s. FB5 is a murine IgG1 antibody that binds to endosialin and has been shown to recognize endothelial cells associated with a variety of different cancer types. Structural evaluation classified endosialin as a C-type lectin-like protein composed of five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats). The protein also contains a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. The protein appears to be a glycoprotein. Carbohydrate analysis shows that the endosialin core protein has an abundance of sialylated, O-linked oligosaccharides with similarities to sialomucin-like molecules. Subsequent work combined the complementarity determining regions (CDR) of the mouse FB5 onto a human IgG1 backbone to create a humanized antibody that is able to also bind to vessels within malignant tissues and a subset of cells in HMVEC cultures. U.S. Pat. No. 5,342,757 describes the FB5 antibody that binds to a ˜100 kDa protein.
Endosialin offers an opportunity to specifically target cancer and neovascular disease. Provided herein are in-out antibodies that can, in the alternative, internalize in endosialin-positive cells (e.g., for delivery of toxic conjugates) and elicit a cytotoxic effect via immune effector activity. The antibodies of the invention provide effective antibody therapies for cancer, neovascular-associated diseases such as but not limited to macular degeneration (e.g., age-related macular degeneration), retinopathy, and inflammation, and other conditions associated with endosialin.